Oligonucleotide labeling reactants and their use

ABSTRACT

The invention relates to a novel labeling reactant of formula (I) suitable for labeling an oligonucleotide  
                 
 
     wherein:  
     R is a temporary protecting group. A is either a phosphorylating moiety or a solid support tethered to a bridge point Z via a linker arm E. E′ is a linker arm between G and Z. G is a bivalent aromatic structure, tethered to two iminodiacetic acid ester groups N(COOR′″) 2  or G is a structure selected from a group consisting of  
                 
 
      or  
     G is a protected functional group. The invention further concerns a method for direct attachment of a conjugate group to an oligonucleotide structure enabling the attachment of a desired number of these groups during chain assembly. The method comprises a Mitsonobu alkylation.

FIELD OF THE INVENTION

[0001] This invention relates to novel compounds and methods forlabeling of oligonucleotides using machine assisted solid phasechemistry.

BACKGROUND OF THE INVENTION

[0002] The publications and other materials used herein to illuminatethe background of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference.

[0003] Synthetic oligonucleotides tethered to various ligands have beenused as research tools in molecular biology [see e.g.: Goodchild,Bioconjugate Chem., 1990, 3, 166; Uhlman and Peyman, Chem. Rev., 1990,90, 543; Sigman, et al. Chem. Rev., 1993, 93, 2295; O'Donnel andMcLaughlin in Bioorganic Chemistry, Nucleic Acids, Hecht SM, ed. OxfordUniv. Press, 1996, p. 216]. They have been applied to genetic analysis,and to elucidate mechanism of gene function. Oligonucleotides carryingreporter groups have had widespread use for automated DNA sequencing,hybridization affinity chromatography and fluorescence microscopy.Oligonucleotide-biotin conjugates are widely used as hybridizationprobes. Antisense oligonucleotides covalently linked to intercalators,chain cleaving or alkylating agents have been shown to be efficient asgene expression regulators. The sequence specific artificial nucleases,when targeted against mRNA, may find applications even aschemotherapeutics.

[0004] For several applications, such as in DNA hybridization assays, itis desirable to introduce more than one reporter group to theoligonucleotide structure. This can be performed by three alternativemethods:

[0005] (i) by coupling several base- or carbohydrate-tetherednucleosidic building blocks to the growing oligonucleotide chain,

[0006] (ii) by functionalization of the internucleosidic phosphodiesterlinkages, or

[0007] (iii) by using several multifunctional non-nucleosidic buildingblocks during the oligonucleotide chain assembly.

[0008] All of these methods have their own drawbacks. Since the doublehelix formation of DNA is based on hydrogen bonding between thecomplementary base residues, tethers attached to the base moieties oftenweaken these interactions. This problem is easily overcome by using thetethered nucleosides at the 3′- or 5′-terminus of the coding sequence,or by using labels linked to C5 of pyrimidine residues. Introduction oftethers to the phosphate backbone gives rise to new chiral centers andmakes the purification of these analogues difficult. Introduction of thetether arm to the carbohydrate moiety, in turn, often decreases thecoupling efficiency of the phosphoramidite (steric hindrance).Furthermore, synthesis of these blocks is commonly extremely laborious.Although design of non-nucleosidic blocks may look attractive on paper,very often their syntheses suffer from complexity, low coupling yieldsand problems associated with the storage and handling of thephosphoramidites. For commercial applications design of base tetherednucleosidic building blocks is often the method of choice.

[0009] Introduction of linker arms to the nucleobase is most commonlyperformed by allowing a nucleoside with a good leaving group (N-tosyl,N-benzoyl, halogen, triazole, thiol) at C4 of pyrimidines or C2, C8 orC6 of purines to react with the appropriate nucleophilic linker molecule(e.g. an alkane-α,ω-diamine). Since normally an excess of linkermolecule and rather vigorous reaction conditions has to be used,laborious purification procedures cannot bc avoided. The basic reactionconditions needed gives additional requirements to the protecting groupsin the target molecule. These problems may be overcome by attachment ofthe linker molecules to C5 of pyrimidine bases by a palladium catalyzedcoupling reaction between 5-halogeno pyrimidine 5-mercuriochloronucleoside and an alkynyl or allyl linker, respectively. However, themethod involves rather laborious synthesis of a 5-halogeno or5-mercuriochloro nucleoside. Very recently, attachment of a linker armto the N3 of 3′,5′-O-protected thymidine based on Mitsunobu reaction[Mitsunobu, Synthesis, 1981, 1] was reported [J. Org. Chem., 1999, 64,5083; Nucleosides, Nucleotides, 1999, 18, 1339]. Since the couplingreaction is performed under mild conditions, a wide range of tether armscan be introduced.

[0010] Most of the methods for oligonucleotide tethering described inliterature involves attachment of functional groups in theoligonucleotide structure during chain assembly. Hence, introduction ofthe label molecules has to be performed in solution. In the labelingreaction the additional amino or mercapto groups of oligonucleotides areallowed to react in solution with isothiocyanato, haloacetyl or2,4,6-triazinyl derivatives of label molecules. Carboxylic acid groups,in turn, can be labeled with amino tethered labels with the aid ofwater-soluble carbodiimide. Since in all the cases the labeling reactionis performed in aqueous solution with an excess of labeling reactants,laborious purification procedures cannot be avoided. Especially whenattachment of several labels is required the isolation andcharacterization of the desired conjugate is extremely difficult, andoften practically impossible. Hence, several attempts to incorporatelabel molecules or their appropriately protected precursor tooligonucleotide structure during chain assembly have been done [Ruth, JL et al, U.S. Pat. No. 4,948,882; Brush, C K et al, U.S. Pat. No.5,583,236]. The fluorescent label monomers for solid phase chemistrysynthesized are most commonly organic dyes (e.g. fluorescein, rhodamine,dansyl, dabsyl, pyrene, TAMRA) several of these are even commerciallyavailable. However, such labels and labeled biomolecules suffer frommany commonly known drawbacks such as Raman scattering, otherfluorescent impurities, low water solubility, concentration quenchingetc. In the specific binding assays, generally very low concentrationsof analytes to be measured are present. Thus multilabeling ofoligonucleotides with organic fluorophores may not enough enhancedetection sensitivity needed in several applications. For these types ofapplications lanthanide(III) chelates are labels of choice since they donot suffer from this phenomenon. In DNA hybridization assays,time-resolved luminescence spectroscopy using lanthanide chelates iswell known [Hemmilä et al. Bioanalytical Applications of LabellingTechnologies, Wallac Oy, 1994]. Therefore, a number of attempts havebeen made to develop non-luminescent (DELFIA®) and new highlyluminescent chelate labels suitable for time-resolved fluorometricapplications. Many patent publications disclose non-luminescent labels[e.g. EP 0064484 A2, EP 0139675 B1, EP 0298939 A1, U.S. Pat. No.4,808,541 and U.S. Pat. No. 4,565,790]. Highly luminescent labelsinclude e.g. stabile chelates composed of derivatives of pyridines [U.S.Pat. No. 4,920,195, U.S. Pat. No. 4,801,722, U.S. Pat. No. 4,761,481, WO93/11433, U.S. Pat. No. 4,459,186, EP 0770610 A1 and Remuinan et al, J.Chem. Soc. Perkin Trans 2, 1993, 1099], bipyridines [U.S. Pat. No.5,216,134], terpyridines [U.S. Pat. No. 4,859,777, U.S. Pat. No.5,202,423 and U.S. Pat. No. 5,324,825] or various phenolic compounds[U.S. Pat. No. 4,670,572, U.S. Pat. No. 4,794,191 and Ital. Pat. 42508A789] as the energy mediating groups and polycarboxylic acids aschelating parts. In addition, various dicarboxylate derivatives [U.S.Pat. No. 5,032,677, U.S. Pat. No. 5,055,578 and U.S. Pat. No. 4,772,563]macrocyclic cryptates [U.S. Pat. No. 4,927,923, WO 93/5049 and EP0493745 A1] and macrocyclic Schiff bases [EP 369000 A] have beenpatented. Also a method for labeling of biospecific binding reactantssuch as hapten, a peptide, a receptor ligand, a drug or PNA oligomerwith luminescent labels by using solid-phase synthesis has beenpublished [EP 067205A1]. One such oligonucleotide labeling reagent hasbeen synthesized and used in multilabeling of oligonucleotides[Kwiatkowski et al. Nucleic Acids Res., 22, 1994, 2604]. However thesynthetic strategy described allows only preparation of chelates wherethe nucleobase is conjugated to the chelate structure limiting thechelate stability and versatility. Furthermore, the structuresynthesized is usable only with europium(III) but not with terbium(III),dysprosium(III) or samarium(III).

[0011] For some special applications such as helicase assays based onfluorescence energy transfer [Earnshaw et. al. J. Biomol. Screening, 4,1999, 239] large quantities of ultrapure oligonucleotides bearing aluminescent lanthanide(III) chelate at their 3′- or 5′-terminus areneeded. Although these molecules can be obtained by classical labelingmethods in solution, yields of the oligonucleotide conjugates can bedramatically improved and purification procedures can be highlysimplified if the label could be attached to the oligonucleotidestructure during chain assembly. For 5′-derivatization synthesis ofnucleosidic or non-nucleosidic building blocks are needed, while3′-labeling calls for appropriately derivatized polymeric solidsupports.

OBJECTS AND SUMMARY OF THE INVENTION

[0012] The main objective of the present invention is to improvelabeling of oligonucleotides with a desired number of lanthanide(III)chelates.

[0013] One objective of the invention is to provide improved labelingreactants for labeling an oligonucleotide.

[0014] Another objective of the invention is to provide a highlysimplified method for the preparation of nucleosidic building blocksthat allow large-scale preparation of oligonucleotide conjugatescontaining additional functional groups in their structure.

[0015] The invention provides improved labeling reactants and aversatile method for direct attachment of a desired number of conjugategroups to the oligonucleotide structure during chain assembly. Hencesolution phase labeling and laborious purification procedures can beavoided. The key reaction in the synthetic strategy towards nucleosidicoligonucleotide building blocks is a Mitsunobu alkylation which allowsintroduction of various labeling reactants to the nucleoside, andfinally to the oligonucleotide structure. When oligonucleotides labeledwith lanthanide(III) chelates are synthesized, initially precursors oflanthanide(III) chelates are introduced to the oligonucleotide structureduring chain assembly, and they are converted to the correspondinglanthanide(III) chelates during deprotection steps.

[0016] For some applications, e.g. for helicase assays, ultrapureoligonucleotides bearing a single label molecule at 3′- or 5′-terminusare needed. The present approach for the introduction of lanthanide(III)chelates at these positions on solid phase is also demonstrated.

[0017] Thus, the present invention concerns a labeling reactant offormula (I) suitable for labeling an oligonucleotide.

[0018] Wherein:

[0019] R is a temporary protecting group such as 4,4′dimethoxytrityl(DMTr), 4-methoxytrityl (MMTr), trityl (Tr), (9-phenyl)xanthen-9-yl(pixyl) or not present.

[0020] A is either a phosphorylating moiety

[0021]  where

[0022] L is O, S, or not present

[0023] L′ is H, L′″CH₂CH₂CN or L′″Ar, where Ar is phenyl or itssubstituted derivative, where the substituent is nitro or chlorine, andL′″ is O or S;

[0024] L″ is O⁻, S⁻, Cl, N(i-Pr)₂; or

[0025] A is a solid support tethered to Z via a linker arm, which isformed of one to ten moieties, each moiety being selected from a groupconsisting of phenylene, alkylene containing 1-12 carbon atoms,ethynediyl (—C≡C—), ether (—O—), thioether (—S—), amide (—CO—NH—,—NH—CO—, —CO—NR′— and —NR′—CO—), carbonyl (—CO—), ester (—COO— and—OOC—), disulfide (—S—S—), diaza (—N═N—), and tertiary amine (—N—R′),wherein R′ represents an alkyl containing less than 5 carbon atoms.

[0026] Z is a bridge point and is formed from

[0027]  or trivalent derivatives, substituted or unsubstituted, ofcyclohexane, cyclohexene, cyclohexadiene, phenyl, cyclopentane,cyclopentene, cyclopentadiene, cyclobutane, cyclobutene, cyclobutadiene,aziridine, diaziridine, oxetane, thietaneazete, azetidine,1,2-dihydro-1,2-diazete, 1,2-diazetidine, furan, tetrahydrofuran,thiophene, 2,5-dihydrothiophene, thiolane, selenophene, pyrrole,pyrrolidine, phosphole, 1,3-dioxolane, 1,2-dithiole, 1,2-thiolane,1,3-dithiole, 1,3-dithiolane, oxazole, 4,5-dihydrooxazole, isoxazole,4,5-dihydoisozaole, 2,3-dihydroisoxazole, thiazole, isothiazole,imidazole, imidazolidine, pyrazole, 4,5-dihydropyrazole, pyrazolidine,triazole, pyran, pyran-2-one, 3,4-dihydro-2H-pyran, tetrahydropyran,4H-pyran, pyran-4-one, pyridine, pyridone, piperidine, phosphabenzene,1,4-dioxin, 1,4-dithiin, 1,4-oxathiin, oxazine, 1,3-oxazinone,morpholine, 1,3-dioxane, 1,3-dithiane, pyridazine, pyrimidine, pyrazine,piperazine, 1,2,4-triazine, 1,3,5-triazine,1,3,5-triaza-cyclohexane-2,4,6-trione; where

[0028] R″ is H or X′X″, where

[0029] X′ is —O—, —S—, —N—, ON— or —NH— and X″ is a permanent protectiongroup such as t-butyldimethylsilyl-, tetrahydropyranyl,1-(2-fluorophenyl)-4-methoxypiperidin-4-yl-,1-[2-chloro-4-methyl)phenyl]-4-metoxypiperidin-4-yl-,4-methoxytetrahydropyran-4-yl-, pthaloyl-, acetyl, pivaloyl-, benzoyl-,4-methylbenzoyl, benzyl-, trityl or

[0030] X′ is —O— and X″ is alkyl or alkoxyalkylalkyl;

[0031] X is H, alkyl, alkynyl, allyl, Cl, Br, I, F, S, O, NHCOCH(CH₃)₂,NHCOCH₃, NHCOPh, SPh₃, OCOCH₃ or OCOPh.

[0032] E is a linker arm between R and Z, and is formed of one to tenmoieties, each moiety being selected from a group consisting ofphenylene, alkylene containing 1-12 carbon atoms, ethynediyl (—C≡C—),ether (—O—), thioether (—S—), amide (—CO—NH—, —NH—CO—, —CO—NR′— and—NR′—CO—), carbonyl (—CO—), ester (—COO— and —OOC—), disulfide (—S—S—),diaza (—N═N—), and tertiary amine (—N—R′), wherein R represents an alkylcontaining less than 5 carbon atoms, or not present.

[0033] E′ is a linker arm between G and Z, and is formed of one to tenmoieties, each moiety being selected from a group consisting ofphenylene, alkylene containing 1-12 carbon atoms, ethynediyl (—C≡C—),ether (—O—), thioether (—S—), amide (—CO—NH—, —NH—CO—, —CO—NR′— and—NR′—CO—), carbonyl (—CO—), ester (—COO— and —OOC—), disulfide (—S—S—),diaza (—N═N—), and tertiary amine (—N—R′), wherein R′ represents analkyl containing less than 5 carbon atoms, or not present.

[0034] G is a bivalent aromatic structure, tethered to two iminodiaceticacid ester groups N(COOR′″)₂, where

[0035] R′″ is an alkyl of 1 to 4 carbon atoms, allyl,ethyltrimethylsilyl, phenyl or benzyl, which phenyl or benzyl can besubstituted or unsubstituted, and

[0036] said bivalent aromatic structure is capable of absorbing light orenergy and transferring the excitation energy to a lanthanide ion afterthe solid phase synthesis made labeling reactant has been released fromthe used solid support, deprotected and converted to a lanthanidechelate, or

[0037] G is a structure selected from a group consisting of

[0038]  where

[0039] R′″ is an alkyl of 1 to 4 carbon atoms, allyl,ethyltrimethylsilyl, phenyl or benzyl, which phenyl or benzyl can besubstituted or unsubstituted, and one of the hydrogen atoms issubstituted with E′, or

[0040] G is a protected functional group, where the functional group isamino, aminooxy, carboxyl, thiol, and the protecting group is pthaloyl,trityl, 2-(4-nitrophenyl-sulfonyl)ethoxycarbonyl,fluorenylmethyloxycarbonyl, benzyloxycarbonyl or t-butoxycarbonyl foramino and aminooxy, alkyl for carbonyl and alkyl or trityl for thiolprovided that bridge point Z is selected from a group consisting of

[0041] The present invention further concerns a method for directattachment of a conjugate group to an oligonucleotide structure enablingthe attachment of a desired number of these groups during chainassembly. Said method comprises a Mitsunobu alkylation of a compound offormula (II).

R-Z′  (II)

[0042] Wherein:

[0043] R is a temporary protecting group such as DMTr, MMTr, Tr, orpixyl.

[0044] Z′ is an acidic bridge point selected from a group consisting of

[0045] where

[0046] R″ is H or X′X″, where X′ is —O—, —S—, —N—, ON— or —NH— and X″ isa permanent protection group such as t-butyldimethylsilyl-,tetrahydropyranyl, 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl-,1-[2-chloro-4-methyl)phenyl]-4-metoxypiperidin-4-yl-,4-methoxytetrahydropyran-4-yl-, pthaloyl-, acetyl, pivaloyl-, benzoyl-,4-methylbenzoyl, benzyl-, trityl or alkyl;

[0047] X is H, alkyl, alkynyl, allyl, Cl, Br, I, F, S, O, NHCOCH(CH₃)₂,NHCOCH₃, NHCOPh, SPh₃, OCOCH₃ or OCOPh;

[0048] and pK_(a) of said acidic bridge point is <14.

[0049] Said compound of formula (II) is alkylated with a compound offormula(III).

G-E″  (III)

[0050] Wherein:

[0051] E″ is an arm with a primary aliphatic OH group at the end, whicharm is formed of one to ten moieties, each moiety being selected from agroup consisting of phenylene, alkylene containing 1-12 carbon atoms,ethynediyl (—C≡C—), ether (—O—), thioether (—S—), amide (—CO—NH—,—NH—CO—, —CO—NR′— and —NR′—CO—), carbonyl (—CO—), ester (—COO— and—OOC—), disulfide (—S—S—), diaza (—N═N—), and tertiary amine (—N—R′),wherein R′ represents an alkyl containing less than 5 carbon atoms.

[0052] G is a bivalent aromatic structure, tethered to two iminodiaceticacid ester groups N(COOR′″)₂, where

[0053] R′″ is an alkyl of 1 to 4 carbon atoms, allyl,ethyltrimethylsilyl, phenyl or benzyl, which phenyl or benzyl can besubstituted or unsubstituted and

[0054] said bivalent aromatic structure is capable of absorbing light orenergy and transferring the excitation energy to a lanthanide ion afterthe solid phase synthesis made labeling reactant has been released fromthe used solid support, deprotected and converted to a lanthanidechelate, or

[0055] G is a structure selected from a group consisting of

[0056]  where

[0057] R′″ is an alkyl of 1 to 4 carbon atoms, allyl,ethyltrimethylsilyl, phenyl or benzyl, which phenyl or benzyl can besubstituted or unsubstituted, and one of the hydrogen atoms issubstituted with E′, or

[0058] G is a protected functional group, where the functional group isamino, aminooxy, carboxyl, thiol, and the protecting group is pthaloyl,trityl, 2-(4-nitrophenyl-sulfonyl)ethoxycarbonyl,fluorenylmethyloxycarbonyl, benzyloxycarbonyl or t-butoxycarbonyl foramino and aminooxy, alkyl for carbonyl and alkyl or trityl for thiol, or

[0059] G is not present.

[0060] The functional groups of E′ and G, excluding said primaryaliphatic OH group, are protected.

[0061] A compound of formula (IV)

[0062] is produced.

[0063] Wherein:

[0064] G and R of compound (IV) are as defined above;

[0065] E′″ is a linker arm between G and Z, and is formed of one to tenmoieties, each moiety being selected from a group consisting ofphenylene, alkylene containing 1-12 carbon atoms, ethynediyl (—C≡C—),ether (—O—), thioether (—S—), amide (—CO—NH—, —NH—CO—, —CO—NR′— and—NR′—CO—), carbonyl (—CO—), ester (—COO— and —OOC—), disulfide (—S—S—),diaza (—N═N—), and tertiary amine (—N—R′), wherein R′ represents analkyl containing less than 5 carbon atoms; and

[0066] Z″ is a bridge point selected from a group consisting of

[0067]  where

[0068] R″ is H or X′X″, where X′ is —O—, —S—, —N—, ON— or —NH— and X″ isa permanent protection group such as t-butyldimethylsilyl-,tetrahydropyranyl, 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl-,1-[2-chloro-4-methyl)phenyl]-4-metoxypiperidin-4-yl-,4-methoxytetrahydropyran-4-yl-, pthaloyl-, acetyl, pivaloyl-, benzoyl-,4-methylbenzoyl, benzyl-, trityl or alkyl;

[0069] X is H, alkyl, alkynyl, allyl, Cl, Br, I, F, S, O, NHCOCH(CH₃)₂,NHCOCH₃, NHCOPh, SPh₃, OCOCH₃ or OCOPh.

[0070] Advantages and Key Steps of Method for OligonucleotideDerivatization

[0071] The present invention for oligonucleotide derivatization combinesseveral important features:

[0072] (i) The nucleosidic protected functional group tethered buildingblocks can be synthesized in a few days using cheap reagents, equimolarreagent ratios, and simple purification procedures. The startingmaterials are commercially available and can also be prepared in asingle step using standard well-documented textbook protocols [Gait, M.Oligonucleotide Synthesis, a Practical Approach, IRL Press, 1990]. Thekey reaction in the present invention is the Mitsunobu alkylation of theabove mentioned 5′-O-protected nucleoside and the appropriate linkermolecule i.e. a primary alcohol where additional functional groups areprotected. Under the reaction conditions employed 3′-O-protection of thenucleoside is not required. These nucleosides are finally converted tothe corresponding phosphoramidites in conventional manner, and they canbe purified either by precipitation from cold hexanes, or by silica gelcolumn chromatography. Since the products are solids, their storage andhandling does not suffer from the problems associated with oilynon-nucleosidic phosphoramidites.

[0073] (ii) Since the coupling reaction between the nucleoside and thetether molecule is performed under mild reaction conditions [at ambienttemperature in dry tetrahydrofuran (THF)] using equimolar reagentratios, a wide range of tethers can be introduced. The only requirementis that the tether molecule has a primary hydroxyl group in itsstructure, and other functional groups are protected. Hence verycomplicated molecules can be incorporated to the nucleoside (and finallyto the oligonucleotide structure) in high efficiency. These tethers withconjugate groups for different applications can be:

[0074] (a) fluorescent or chemiluminescent groups or spin-labels.

[0075] (b) chemically reactive groups that induce irreversible reactionsto their target sequences, or

[0076] (c) groups that promote intermolecular interactions (e.g.biotin).

[0077]  Representative structures synthesized according to the method ofthe present invention are presented in schemes 2-11.

[0078] (iii) Since the building blocks are derivatives of nucleosidesbearing tether arm attached to the base moiety, they can be coupled tothe oligonucleotide chain using standard protocols in high efficiency(i.e. no changes in concentrations or coupling times required).

[0079] (iv) Since the tether arm is attached to the base moiety,multilabeling of oligonucleotides is achievable.

[0080] (v) If a ligand structure/structures is/are incorporated to theoligonucleotide chain during chain assembly, it/they can be converted tothe corresponding lanthanide(III) chelates during slightly modifieddeprotection steps. Hence laborious solution phase labeling as well assynthesis of the activated chelates and oligonucleotides tethered tofunctional groups can be avoided.

[0081] (vi) For several applications introduction of only a single labelmolecule at the 5′-terminus of the oligonucleotide structure is needed.For these applications the ligand structures can be simplified byomitting the nucleobase from the structure i.e. resulting innon-nucleosidic phosphoramidite building blocks. Examples of such amolecules are shown in examples 22 and 33.

[0082] (vii) For the preparation of 3′-tethered oligonucleotides theligand structures can be converted also to the correspondingnon-nucleosidic or nucleosidic solid supports that can be used in solidphase oligonucleotide synthesis. The solid support can be either a longchain alkylamine controlled pore glass (LCAA) or polystyrene. An exampleof such a solid support is shown in example 24.

[0083] (viii) Several of the structures described above can be obtainedalso by using slightly modified reaction routes:

[0084] (a) A nucleoside tethered to an alkynyl group is synthesized byMitsunobu alkylation, the ligand structure is coupled to it as anaromatic halide using Sonagoshira reaction.

[0085] (b) A nucleoside tethered to a protected functional group issynthesized using Mitsunobu reaction, the protecting group isselectively removed (e.g. ammonolysis for trifluoroacetylamido), and theligand or label structure is coupled by carbodiimide assisted reaction.

DETAILED DESCRIPTION OF THE INVENTION

[0086] The novel labeling reactants and labeling methods of the presentinvention are particularly suitable for the preparation ofoligonucleotide conjugates bearing a desired known number of functionalgroups or label molecules in their structure.

[0087] The term ‘bivalent’ in the definition of G shall mean a chemicalgroup bound to two neighboring atoms.

[0088] The functional groups most suitable are amino, carboxyl, aminooxyor thiol.

[0089] The most suitable chelates are non-luminescent and luminescentlanthanide(III) chelates.

[0090] The organic dyes suitable for monolabeling are dabsyl, dansyl,fluorescein, rhodamine or TAMRA.

[0091] A particularly preferable transient protecting group R is4,4′-dimethoxytrityl

[0092] The sugar of the nucleotide is preferably ribose or2-deoxyribose. In the former case the permanent protecting group X″ forhydroxyl is preferably t-butyldimethylsilyl, tetrahydropyranyl,1-(2-fluorophenyl)-4-methoxypiperidin-4-yl-(Fpmp),1-[2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl- or4-methoxytetrahydropyran-4-yl-, or X″ is an alkyl or alkoxalkyl,preferably methyl, methoxymethyl or etoxymethyl.

[0093] For luminescent labeling reactants G is a bivalent aromaticstructure and is preferably selected from a group consisting ofcarbostyryl or structures disclosed in Scheme 1A. For non-luminescentlabeling reactants G is selected from a group of structures disclosed in1B.

[0094] The substituent R′″ is preferably methyl, ethyl or allyl.

[0095] Most preferably, the labeling reactant is

[0096] 2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-N3{tetramethyl2,2′,2″,2′″-[(4-(1-hexyn-5-yl)pyridine-2,6-diyl)bis(methylennenitrilo)}tetrakis(acetato)uridine3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite (7),

[0097]N3-[6-[4-(dimethylamino)azobenzene-4′-sulfonamido]hex-1-yl-5′-O-(4,4′-dimethoxytrityl)thymidine3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite (12),

[0098]5′-O-(4,4′-dimethoxytrityl)-N3-{tetramethyl-2,2′,2″,2′″-{6,6′-[4′-hydroxyethoxyethoxyphenylethynyl]pyridine-2,6-diyl}bis(methylenenitrilo)tetrakis-(acetato)}thymidine3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite (18),

[0099]tetramethyl-2,2′,2″,2′″-{{6,6′-[4-(6-hydroxyhexyl)-1H-pyrazol-1,3-diyl]bis-(pyridine)-2,2′-diyl}bis(methylenenitrilo)}tetrakis(acetato)-6-O-(2-cyanoethyl)N,N-diisopropyl)phosphoramidite(25),

[0100]2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-3-6-{{4-{6,6″-bis[N,N-bis(methoxycarbonylmethyl)aminomethyl]-2,2′:6′,2″-terpyridine-4′-yl}phenyl}hex-5-yn-1-yl}-uridine3′-[O-(2-cyanoethyl)-N,N-diisopropyl]phosphoramidite (37) or

[0101]6-{4-{6,6″-bis[N,N-bis(methoxycarbonylmethyl)aminomethyl]-2,2′:6′,2″-terpyridine-4′-yl}phenyl}hex-5-yn-1-ol[O-(2-cyanoethyl)-N,N-diisopropyl]-phosphoramidite(38).

[0102] Most preferably the solid support is5′-O-(4,4′-dimethoxytrityl)-3′-O-succinyl-N3-{tetramethyl-2,2′,2″,2′″-{6,6′-[4′-hydroxyethoxyethoxyphenylethynyl]pyridine-2,6-diyl}bis(methylenenitrilo)tetrakis(acetato)}thymidinelong chain alkylamine controlled pore glass (24).

[0103] According to a preferred embodiment the lanthanide chelate is aeuropium(III), terbium(III), samarium(III) or dysprosium(III) chelate.

[0104] The invention is further elucidated by the following examples.The structures and synthetic routes employed in the experimental partare depicted in schemes 2-9. Scheme 2 illustrates the synthesis of thelabeling reagents 3 and 4. The experimental details are given inexamples 1-4. Schemes 3A and 3B illustrate the synthesis of the labelingreagent 8. Scheme 4 illustrates synthesis of the labeling reagent 12.The experimental details are given in examples 10-12. Schemes 5A and 5Billustrate the preparation of the labeling reagent 18. Experimentaldetails are given in examples 13-16. Scheme 6A and 6B illustrate thesynthesis of labeling reagent 25. Experimental details are given inexamples 18-22. Scheme 7A and 7B illustrate the synthesis of the solidsupport 27. Experimental details are presented in examples 23 and 24.Scheme 8A and 8B illustrate the synthesis of labeling reagents 37 and38. Experimental details are given in examples 25-33. Scheme 9illustrates the introduction of primary amino groups to theoligonucleotide structure in the aid of compound 8 as well as furtheroligonucleotide derivatization in solution. Experimental details aregiven in example 35. Scheme 10 illustrates introduction oflanthanide(III) chelates to the oligonucletide structure in with the aidof compound 8. Experimental details are given in example 36. Scheme 11illustrates introduction of lanthanide(III) chelates to theoligonucleotide structure in with the aid of compound 38. Experimentaldetails are given in example 37.

EXPERIMENTAL PROCEDURES

[0105] Reagents for machine assisted oligonucleotide synthesis werepurchased from PE Biosystems (Foster City, Calif.).2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphodiamidite,N6-trifluoroacetamidohexanol and2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-uridine and5′-O-(4,4′-dimethoxy-trityl)thymidine were synthesized according topublished procedures. Adsorption column chromatography was performed oncolumns packed with silica gel 60 (Merck). NMR spectra were recorded ona Jeol LA-400 spectrometer operating at 399.8, 350, 161.9 and 100.5 MHzfor ¹H, ¹⁹F, ³¹P and ¹³C, respectively, or on a Jeol GX 500 instrumentoperating at 500.00 and 125.65 MHz for ¹H and ¹³C, respectively. Me₄Siwas used as an internal (¹H and ¹³C) and H₃PO₄ (³¹P) and trifluoroaceticacid (¹⁹F) as external references. Coupling constants are given in Hz.When reported, signal characterization is based on ¹H, ¹H, ¹H, ¹³C and¹³C, ¹³C COSY experiments. IR spectra were recorded on a Perkin Elmer2000 FT-IR spectrophotometer. Fast atom bombardment mass spectra wererecorded on a VG ZabSpec-ao TOF instrument in the positive detectionmode. Oligonucleotides were assembled on an Applied Biosystems 932 DNASynthesizer using phosphoramidite chemistry and recommended protocols(DMTr-Off-synthesis).

Example 1

[0106] The synthesis of2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-N3-(N6-trifluoroacetamidohex-1-yl)uridine(1)

[0107] 2′-Deoxy-5′-O-(4,4′-dimethoxytrityl)uridine (8.0 g, 15.1 mmol),Ph₃P (4.7 g, 17.9 mmol) and N6-trifluoroacetamidohexan-1-ol (4.1 g, 18.1mmol) were dissolved in dry THF (80 ml). DEAD (2.85 ml) was added infive portions during 15 min, after which the mixture was stirred 2 h atambient temperature and concentrated. Purification on silica gel (eluentdiethyl ether) yielded 65% of 2. ¹H NMR (DMSO-d₆: 500 MHz); δ 9.41 (1H,br, NH); 7.72 (1H, d, H-6); 7.35 (2H, DMTr); 7.25 (7H, DMTr); 6.85 (4H,d, DMTr); 5.5 (1H, d, H-5), 6.2 (1H, t, H-1′), 5.4 (1H, d, 3′-OH), 4.3(1H, m, H-4′), 4.3 (1H, m, H-3′), 3.9 (1H, m, H-4′), 3.5 (1H, dd, H-5′),3.8 (2H, t), 3.2 (1H, H-5″), 3.15 (2H, m), 2.2 (2H, H-2′; H-2″), 1.5(4H, m); 1.25 (4H, m). ¹³C NMR (DMSO-d₆): δ 161.7 (C4), 158.0 (C═O),156.5 (q, CF₃); 150.3 (C2); 144.8 (DMT); 138.8 (C6); 129.7, 127.8,127.7, 126.7, 113.1 (DMT); 100.7 (C-5), 85.7 (DMT); 85.5 (C4′); 85.2(C1′); 69.8 (C3′); 63.3 (C5′); 55.5 (2.OMe); 40.1 (NCH₂); 39.7 (C2′);39.0 (CH₂NHCO); 28.0, 25.9, 25.8 (CH₂) ¹⁵N NMR (DMSO-₆): δ-294.5(NHCOCF₃); -264.0 (N1); -244.6 (N3).

Example 2

[0108] The synthesis of5′-O-(4,4′-dimethoxytrityl)-N3-(N6-trifluoroacetamidohexyl)-thymidine(2)

[0109] The title compound was synthesized as described in example 1 forcompound 1 by using 5′-O-(4,4′-dimethoxytrityl)thymidine as the startingmaterial. The yield was 76%. ¹H NMR (DMSO-d₆; 500 MHz): δ 9.35 (1H, brt, J 5.2, NH); 7.54 (1H, d, J 1.1, H-6); 7.38-7.23 (9H, DMT); 6.88 (4H,d, DMT); 6.22 (1H, t, J 6.6, H-1′); 5.31 (1H, d, J 4.6, 3′-OH); 4.31(1H, m, H-3′); 3.89 (1H, m, H-4′); 3.77 (2H, m, NCH₂); 3.72 (6H, s,2.OCH₃); 3.21 (1H, dd, J 5.8 and 10.6H-5′); 3.16 (1H, dd, J 3.0 and10.6, H-5″); 3.15 (2H, m, CH₂NH); 2.24 (1H, m, H-2″); 2.17 81H, m,H-2′); 1.49 (3H, d, J 1.1 5-CH₃); 1.48 (2H, m, NCH₂CH₂); 1.45 (2H, m,CH₂CH₂NH); 1.26 (4H, m, 2.CH₂). ¹³C NMR (DMSO-d₆) δ: 162.5 (C4), 158.1(C═O), 156.1 (q, J_(C,F) 35.9, CF₃); 150.2 (C2); 144.7 (DMT); 134.3(C6); 129.7, 127.8, 127.66, 126.7, 113.1 (DMT); 108.7 (C-5), 85.7 (DMT);85.6 (C4′); 84.8 (C1′); 70.4 (C3′); 63.7 (C5′); 55.0 (2.OMe); 40.4(NCH₂); 39.7 (C2′); 39.0 (CH₂NH—CO); 28.0, 26.9, 25.9, 25.8 (CH₂).

Example 3

[0110] The synthesis of2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-N3-(N6-trifluoroacetamidohexyl)uridine3′-O-(2-cyanoetlyl N,N-diisopropyl)phosphoramidite (3)

[0111] Predried compound 1 and 2-cyanoethylN,N,N′,N′-tetraisopropylphosphordiamidite (1.5 eq) were dissolved in dryacetonitrile. 1H tetrazole (1 eq; 0.45 M in acetonitrile) was added, andthe mixture was stirred for 30 min at room temperature before beingpoured into 5% NaHCO₃ and extracted with dichloromethane and dried overNa₂SO₄. Precipitation from cold (−70° C.) hexane yielded the titlecompound as a white powder. Compound 3: ³¹P NMR (CDCl₃): δ 148.6 (0.5P), 148.4 (0.5 P).

Example 4

[0112] The synthesis of5′-O-(4,4′-dimethoxytrityl)-N3-(N6-trifluoroacetamidohexyl)-thymidine3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite (4)

[0113] Phosphitylation of compound 2 as described in example 3 forcompound 1 yielded the title compound as a white powder. Compound 4: ³¹PNMR (CDCl₃): δ 148.6 (0.5 P), 148.4 (0.5 P).

Example 5

[0114] The synthesis of tetramethyl2,2′,2″,2′″-[4-(6-hydroxyhex-5-yn-1-yl)pyridine-2,6-diyl)bis(methylenenitrilo)]tetrakis(acetate)(6)

[0115] A mixture of tetramethyl2,2′,2″,2′″-[4-bromopyridine-2,6-diyl)bis(methylenenitrilo)tetrakis(acetate)(5), bis(triphenylphosphinepalladium(II) chloride and CuI in dry THF andtriethylamine was deaerated with argon. 5-hexynol was added and themixture was stirred for 7 h at 55° C. The cooled solution was filtered;the filtrate was evaporated and redissolved in dichloromethane. Thesolution was washed with water, dried and concentrated. Purification onsilica gel yielded the title compound as an oil (75%). Compound 6: ¹HNMR (CDCl₃; 400 MHz): 7.46 (2H, s); 3.99 (4H, s); 3.71 (12H, s, 4 CH₃);3.62 (8H, s, 4 CH₂); 2.53 (4H, m, CH₂); 1.70 (4H, m, 2 CH₂) IR (neat):2242 (C≡C).

Example 6

[0116] The synthesis of2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-N3{tetramethyl2,2′,2″,2′″-[(4-(hex-5-yn-1-yl)pyridine-2,6-diyl)bis(methylenenitrilo)}tetrakis(acetato)-uridine(7)—Method A

[0117] 2′-Deoxy-5′-O-(4,4′-dimethoxytrityl)uridine was allowed to reactwith compound 6 as described in example 1. Purification on silica gel(eluent CH₂Cl₂:MeOH 95:5, v/v) yielded the title compound as foam. Theyield was 70%. Compound 7: ¹H NMR (DMSO-d₆; 500 MHz): δ 7.67 (1H, d, J8.2, H-6); 7.36 (2H, s, pyridine); 7.35 (2H, DMTr); 7.25 (7H, DMTr);6.85 (4H, d, DMTr); 6.16 (1H, t, H-1, J 6.3); 5.48 (1H, d, J 8.1, H-5);5.34 (1H, d, J 4.8, 3′-OH); 4.29 (1H, m, H-3′); 3.89 (1H, m, H-4′); 3.86(4H, s, 2.CH₂); 3.82 (2H, t, J 5.6 Ar—CH₂); 3.58 (8H, s, 4.CH₂), 3.24(1H, dd, J 10.7 and 5.2, H-5′); 3.19 (1H, dd, J 3.1 and 10.7, H-5″);2.50 (2H, t, CH₂); 2.20 (2H, t, H-2′ and H-2″); 2.72 (1H, br, OH) 1.73(2H, m, CH₂); 1.52 (2H, m, CH₂).

Example 7

[0118] Synthesis of2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-N3(hex-5-yn-1-yl)uridine (9)

[0119] 5-Hexynol was allowed to react with2′-deoxy-5′-O-(4,4′-dimethoxytrityl)uridine under Mitsunobu conditionsdescribed in example 1. Purification on silica gel (eluent diethylether) yielded the title compound as a solid (86%) Compound 9: ¹H NMR(DMSO-d₆, 400 MHz): δ 7.70 (1H, d, J 8.1, H-6); 7.39 (2H, DMT); 7.31(2H, DMT); 7.25 (5H, DMT); 6.90 (4H, d, J 8.0); 6.18 (1H, t, J 6.3,H-1′) 5.49 (1H, J 8.1, H-5); 5.38 (1H-1, d, J 4.6, 3′-OH); 4.31 (1H, m,H-3′); 3.90 (1H, m, H-4′); 3.78 (2H, m, NCH₂); 3.74 (6H, s, 2.OCH₃);3.26 (1H, dd, J 5.4 and 10.7, H-5′); 3.19 (1H, dd, J 2.9 and 10.7,H-5″); 2.23 (3H, H-2′, H-2″ and CH₂C≡); 1.63 (2H, p, CH₂); 1.47 (1H, t,≡CH); 1.43 (2H, p, CH₂).

Example 8

[0120] The synthesis of2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-N3{tetramethyl2,2′,2″,2′″-[(4-(hex-5-yn-1-yl)pyridine-2,6-diyl)bis(methylene-nitrilo))}tetrakis(acetato)-uridine(7)—Method B

[0121] Compound 9 was coupled to compound 5 using the method describedin example 5. The yield was 60%. The product was spectroscopically andchromatographically identical with the material synthesized in example6.

Example 9

[0122] The synthesis of2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-3-{6-{2,6-bis[N,N-bis(methoxycarbonylmethyl)aminomethyl]pyridin-4-yl}hex-5-yn-1-yl}uridine3′-[O-(2-cyanoethyl)-N,N-diisopropyl]phosphoramidite (8)

[0123] Compound 7 was phosphitylated using the method described inexample 2. Purification was performed on silica gel (eluentCH₂Cl₂:Et₃N:MeOH 90:10:5; v/v/v). Compound 8: ³¹P NMR (CDCl₃): δ 149.4(0.5 P), 149.1 (0.5 P).

Example 10

[0124] The synthesis of6-[4-(dimethylamino)azobenzene-4′-sulfonamido]hexan-1-ol (10)

[0125] To a stirred solution of 6-aminohexan-1-ol (0.50 g, 4.27 mmol) indichloromethane (10 ml) was added dropwise a solution of dabsyl chloride(0.5 g, 1.54 mmol) in dichloromethane (10 ml). After 1 h the mixture vaswashed with sat aq. NaHCO₃. The organic layer was dried over Na₂SO₄ andconcentrated. Purification on silica gel (eluent CH₂Cl₂ containing 1%(v/v) MeOH) yielded the title compound as red solid. Compound 10: ¹H NMR(CDCl₃): δ 7.97-7.89 (6H, m); 6.76 (2H, d, J 9.3); 4.50 (1H, br t, J6.3); 3.60 (2H, t, J 6.2); 3.13 (6H, s); 2.98 (2H, q, J 6.9); 1.63 (1H,br); 1.50 (4H, m); 1.30 (4H, m).

Example 11

[0126] The synthesis ofN3-[6-[4-(dimethylamino)azobenzene-4′-sulfonamido]hex-1-yl-5′-O-(4,4′-dimethoxytrityl)thymidine(11)

[0127] The title compound was synthesized by Mitsunobu alkylation of5′-O-(4,4′-dimethoxytrityl)thymidine and compound 10 using proceduresdescribed in example 1. The yield was 74%. Compound 11: ¹H NMR (400 MHz,DMSO-d₆): d 7.98-7.88 (6H, m, dabsyl); 7.57 (1H, J 1.2H-6); 7.40 (2H, d,DMT); 7.30-7.24 (7H, m, DMT); 6.83 (4H, d, J, 9.0, DMT), 6.75 (2H, d, J7.3, dabsyl); 6.48 (1H, dd, J 7.8), 5.03 (1H, t, J 6.1, NH); 4.55 (1H,m, H-3′); 4.07 (1H, m, H-4′); 3.92 (2H, m, NCH₂); 3.79 (6H, s, 2.OCH₃);3.46 (1H, dd, J 3.2 and 10.5, H-5′); 3.36 (1H, dd, J 2.9 and 10.5,H-5″); 3.12 (6H, s, N(CH₃)₂); 2.99 (2H, m, CH₂NH); 2.46 (1H, m, H-2″);2.31 (1H, m, H-2′ and 3′-OH); 1.59 (4H, m, 2.CH₂); 1.38-1.25 (4H, m,2.CH₂).

Example 12

[0128] The synthesis ofN3-[6-[4-(dimethylamino)azobenzene-4′-sulfonamido]hex-1-yl-5′-O-(4,4′-dimethoxytrityl)thymidine3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite (12)

[0129] Phosphitylation of compound 11 as described in example 3 yieldedthe title compound as a solid (purification on silica gel using theeluent described in example 9).

Example 13

[0130] The synthesis of Tritylethoxyethanol (13)

[0131] Bisethoxyethanol (10 ml) was dried by coevaporation with drypyridine and dissolved in the same solvent (20 ml). Trityl chloride wasadded and the mixture was stirred 2 h at ambient temperature. Thesolvent was evaporated in vacuo. The residue was dissolved in methylenechloride, washed with sat. NaHCO₃, dried (Na₂SO₄) and concentrated.Precipitation from ethyl ether yielded the title compound as a whitepowder. It was used in the next step without further characterization.

Example 14

[0132] The synthesis of 4-iodophenoxyethoxyethanol (14)

[0133] Compound 13 was allowed to react with 4-iodophenol as describedin example 1. When the reaction was completed (ca. 2 h) the solvent wasevaporated off and the residue was suspended in diethyl ether and passedthough a short column of silica gel. The eluent was removed in vacuo andthe residue was dissolved in the mixture of TFA and ethanol (9:1, v/v)and stirred overnight at ambient temperature after being concentrated.The residue was taken in methylene chloride and washed with sat. NaHCO₃,dried (Na₂SO₄) and concentrated. Purification was performed on silicagel. The column was eluted initially with methylene chloride to removetrityl carbinol and then with the mixture of CH₂Cl₂:MeOH (9:1, v/v) toelute the desired product. Compound 14: ¹H NMR (CDCl₃): δ 7.53 (2H, d, J9.0); 6.68 (2H, d, J 9.0); 4.07 (2H, m); 3.83 (2H, m); 3.73 (2H, m);3.64 (2H, m); 2.26 (1H, br).

Example 15

[0134] The synthesis of tetramethyl2,2′,2″,2′″-{6,6-[4′-hydroxyethoxyethoxyphenoxy-ethynyl]pyridine-2,6-diyl}bis(methylene-nitrilo)tetrakis(acetate)(16)

[0135] Compound 14 was allowed to react with tetramethyl2,2′,2″,2′″-[4-ethynyl-pyridine-2,6-diyl)bis(methylenenitrilo)tetrakisacetate15 using the reaction described in example 5, but reaction was completedin 5 h at ambient temperature. Compound 15: ¹H NMR (CDCl₃): δ 7.53 (2H,s); 7.49 (2H, d, J 8.8); 6.98 (2H, d, J 8.8); 4.17 (2H, m); 4.03 (4H,s); 3.89 (2H, m); 3.79 (2H, m); 3.72 (12H, s); 3.68 (2H, m); 3.64 (8H,s); 2.38 (1H, br).

Example 16

[0136] The synthesis of5′-O-(4,4′-dimethoxytrityl)-N3-{tetramethyl-2,2′,2″,2′″-{6,6′-[4′-ethoxyethoxyphenoxyethynyl]pyridine-2,6-diyl}bis(methylenenitrilo)-tetrakis(acetato)}thymidine(17)

[0137] Compound 16 was allowed to react with5′-O-(4,4′-dimethoxytrityl)thymidine using the reaction described inexample 1. Compound 17: ¹H NMR (DMSO-d₆): d 7.59 (1H, s); 7.51 (2H, d, J8.8); 7.47 (2H, s); 7.49-7.23 (9H, DMT); 6.99 (2H, d, J 8.8); 6.86(4H,d, DMT); 6.23 (1H, t, J 6.8); 5.34 (1H, d, J 4.2; exch. with D₂O);4.32 (1H, m); 4.10 (2H, m); 4.01 (2H, m); 3.92 (1H, m); 3.90 (4H, s);3.74 (2H, m); 3.71 (12H, s); 3.62 (2H, m); 3.61 (8H, s); 3.10 (2H, m);2.22 (2H, m); 1.47 (3H, s).

Example 17

[0138] The synthesis of2,2′-(4-iodo-1H-pyrazol-1,3-diyl)bis(pyridine)1,1′-dioxide (19)

[0139] To a stirred solution of 2,2′-(1H-pyrazol-1,3-diyl)bis(pyridine)1,1′-dioxide (9.9 g, 38.9 mmol) in conc. nitric acid/water 25 ml (1:1,v/v) iodine (9.8 g, 38.9 mmol) was added and the mixture was heatedovernight at 95° C. The mixture was allowed to cool to room temperatureand alkalized with 1 M NaOH. The aqueous layer was extracted three timeswith CHCl₃/EtOH (4:1), dried (Na₂SO₄) and concentrated. Purification onsilica gel (eluent CH₂Cl₂:MeOH. 9:1, v/v) yielded 12.2 g (82%) of 19. ¹HNMR δ (CDCl₃) 9.52 (1H., s); 8.37 (1H, m); 8.34 (1H, m); 8.09 (1H, m);7.51 (1H, m); 7.41-7.30 (3H, m); 7.25 (1H, m).

Example 18

[0140] The synthesis of6,6′-(4-Iodo-1H-pyrazole-1,3-diyl)bis(pyridine)-2,2′-dicarbonitrile (20)

[0141] Trimethylsilyl cyanide (16.2 ml, 0.13 mol) was added to a mixtureof 19 (4.92 g, 13.0 mmol) and CH₂Cl₂ (133 ml). After 5 min, benzoylchloride (6 ml, 52 mmol) was added, and the mixture was stirred for 24 hat ambient temperature. The mixture was then concentrated (to ca. 15ml), 10% K₂CO₃ solution (130 ml) was added and the mixture stirred for 2h. A cold mixture was filtered, and the main product fraction was washedwith water (50 ml) and cold CH₂Cl₂ (2×20 ml). The organic phase offiltrate was separated, and evaporated to dryness. A cooled mixture ofthe residue and diethyl ether (200 ml) was filtered. Total yield was4.36 g (85%); IR (KBr) 2237 cm⁻¹ (C≡N), 1590, 1574 cm⁻¹ (arom.); ¹H NMRδ (CDCl₃) 8.79 (1H, s); 8.30 (1H, dd, J 0.8 and 3.9); 8.34 (1H, dd, J0.8 and 3.5); 8.02 (1H, dd, J 7.5 and 8.5); 7.95 (1H, dd, J 7.7 and8.1); 7.73 (1H, dd, J 1.0 and 7.7); 7.66 (1H, dd, J 0.8 and 7.5).

Example 19

[0142] The synthesis of tetramethyl2,2′,2″,2′″-{[6,6′-(4-Iodo-1H-pyrazole-1,3-diyl)bis-(pyridine)-2,2′-diyl]bis(methylenenitrilo)}tetrakis(acetate)(22)

[0143] A suspension of compound 20 (5.06 g, 12.7 mmol) and drytetrahydrofuran (140 ml) was deaerated with nitrogen. Borane intetrahydrofuran (1 M, 140 ml) was added within 10 min into the reactionmixture. After stirring for 24 h at room temperature, excess borane wasdestroyed by addition of MeOH, the mixture was evaporated and theresidue dissolved in MeOH saturated with dry HCl (20 ml). After stirringfor 1 h, the mixture was evaporated, and the residue treated withtetrahydrofuran (20 ml). The cooled mixture was filtered and the solidmaterial washed with cold tetrahydrofuran (10 ml) and diethyl ether (5ml). To give 7.47 g (94%) [ms (FAB⁺) 407] of crude compound 21. Amixture of this material (3.1 g, 5.3 mmol), BrCH₂COOMe (2.0 ml, 21.1mmol), dry N,N-diisopropylethylamine (13.8 ml, 79 mmol) and dryacetonitrile (110 ml) was refluxed for 24 h. After evaporation, theresidue was dissolved in CHCl₃ (50 ml), washed with water (3×25 ml) anddried with Na₂SO₄. The product was purified by flash chromatography[silica gel, petroleum ether (40-60′)/ethyl acetate, 1:1]; yield 56% ofcompound 22. IR (film) 1732 cm⁻¹ (C═O), 1144 cm⁻¹ (C—O); ¹H NMR δ(CDCl₃)1.47 (18H, s), 1.48 (18H, s), 3.53 (4H, s), 3.57 (8H, s), 4.06 (2H, s),4.15 (2H, s), 7.54 (1H, d, J=7.6 Hz), 7.72 (1H, d, J=8.0 Hz), 7.79 (1H,t, J=7.6 Hz), 7.82 (1H, t, J=8.0 Hz), 7.91 (1H, d, J=7.6 Hz), 7.95 (1H,d, J=8.0 Hz), 8.70 (1H, s).

Example 20

[0144] The synthesis of tetramethyl2,2′,2″,2′″-{{6,6′-(4-(5-hydroxyhexyn-1-yl)-1H-pyrazole-1,3-diyl)bis(pyridine)-2,2′-diyl}bis(methylenenitrilo)}tetrakis(acetate)(23)

[0145] A mixture of compound 22 (1.0 g, 1.44 mmol), 5-hexyn-1-ol (0.19ml, 1.72 mmol), dry piperidine (4.5 ml) and dry DMF (6 ml) was deaeratedwith argon. Bis(triphenylphosphine)palladium(II) chloride (20 mg, 29μmol) and copper iodide (11 mg, 58 μmol) was added and the mixture wasstirred for 12 h at 40° C. After evaporation, the residue was dissolvedin CHCl₃ (90 ml), washed with water (3×45 ml) and dried with Na₂SO₄. Theproduct was purified by flash chromatography (eluent CH₂Cl₂:MeOH, 9:1).Yield, 0.90 g. [M+H]⁺ 665.

Example 21

[0146] The synthesis of tetramethyl2,2′,2″,2′″-{[6,6′-(4-(hexan-6-ol)-1H-pyrazole-1,3-diyl)bis(pyridine)-2,2′-diyl]bis(methylene-nitrilo)}tetrakis(acetate)(24)

[0147] A mixture of compound 23 (0.45 g, 0.68 mmol) 10% Pd on carbon (50mg) and MeOH (30 ml) was stirred in a hydrogen atmosphere for 2.5 h.After filtration, the filtrate was evaporated and the residue waspurified by flash chromatography (CH2CL2:MeOH, 9:1). The yield was 350mg, 77%; ms 669 [M+H]⁺

Example 22

[0148] The Synthesis of the Non-Nucleosidic Phosphoramidite (25)

[0149] Compound 24 was phosphitylated using the method described inexample 3. Yield after silica gel column chromatography (CH₂Cl₂:MeOH:TEA9:1:1; v/v/v). ³¹P NMR (CDCl₃): 147.8.

Example 23

[0150] The synthesis of5′-O-(4,4′-dimethoxytrityl)-N3-{tetramethyl-2,2′,2″,2′″-{6,6′-[4′-hydroxyethoxyethoxyphenyl-ethynyl]pyridine-2,6-diyl}bis(methylenenitrilo)tetrakis(acetato)}thymidine3′-succinate (26)

[0151] Compound 17 (0.67 mmol) was dissolved in dry pyridine (5 ml).Succinic anhydride (135 mg, 1.35 mmol) and cat. amount of DMAP wereadded, and the mixture was stirred overnight at room temperature andconcentrated. The residue was dissolved in dichloromethane, washed withaqueous triethylamine and dried. Purification was performed on silicagel (eluent: 10% MeOH in dichloromethane). Compound 26: ¹H NMR(DMSO-d₆): δ 7.59 (1H, s); 7.54 (2H, d, J 8.8); 7.50 (2H, s); 7.38-7.21(9H, DMTr); 7.00 (2H, d J 8.8); 6.90 (4H, d, DMTr); 6.27 (1H, dd); 5.31(1H, m); 4.11 (3H, m); 4.02 (2H, m); 3.91 (4H, s); 3.79 (2H, m); 3.73(8H, s); 3.61 (2H, m); 3.60 (12H, s); 3.37 (2H, m); 2.67 (2H, t); 2.42(2H, t); 2.22 (2H, m); 1.47 (3H, s).

Example 24

[0152] The Synthesis of the Solid Support (27)

[0153] Long chain alkylamine controlled pore glass was treated with amixture of 10% TEA in 80% aqueous ethanol, washed with acetonitrile anddried. Compound 23 (0.5 mmol; as a pyridinium salt),N,N′-diisopropylcarbodiimide (1.0 mmol, 157 μl); andN-hydroxysuccinimide (0.5 mmol, 58 mg) was added to a suspension of thesolid support in dry pyridine (5 ml) and the mixture was shakenovernight at ambient temperature. The suspension-was filtered, washedwith dry pyridine, kept in a mixture of Ac₂O:pyridine:N-methylimidazole(1:5:1; v/v/v) for 10 min, and finally washed with ether. Loading asjudged on DMTr cation assay was 34 μmolg⁻¹.

Example 25

[0154] The synthesis of (E)-3-(4″Bromophenyl)-1-pyrid-2′-yl)prop-2-enone(28)

[0155] 4-Bromobenzaldehyde (50 g, 0.27 mol) was added in the ice-coldmixture methanol (540 ml) and water (110 ml) containing potassiumhydroxide (15.2 g). After all aldehyde was dissolved 2-acetylpyridine(30.3 ml, 0.27 mol) was added and the reaction was allowed to proceedovernight at ambient temperature. The precipitation formed was filtered,washed with cold methanol and dried. Yield was 64 g (82%). 1H NMR(CDCl₃): δ 8.75 (1H, br. d); 8.31 (1H, d, J 12 Hz); 8.20 (1H, br d);7.90 (1H, m); 7.87 (1H, d, J 12); 7.59 (5H, m). MS (EI+) 288, 289 [M+].

Example 26

[0156] The synthesis of 4″-(4′″-bromophenyl)-2,2′:6′,2″-terpyridine (30)

[0157] A mixture of compound 28 (20.6 g, 71 mmol), dry ammonium acetate(137 g) and freshly prepared N-[2-(pyrid-2′-yl)-2-oxo-ethyl]pyridiniumiodide (29; 23.3 g, 71 mmol) in dry methanol (650 ml) was heated atreflux overnight. The mixture was cooled to room temperature andrefrigerated. The precipitation was separated by filtration, washed withcold methanol and dried. Yield was 12.5 g (45%). 1H NMR (dmso-d6) δ:8.77 (2H, br d, J 4); 8.71 (2H, s); 8.69 (2H, d J 7.9); 8.06 (2H, td, J2.5 and 7.5); 7.92 (2H, d, J 7.5); 7.79 (2H, d, J 7.5); 7.55 2H, m). MS(EI+) 388, 390 [M+].

Example 27

[0158] The synthesis of 4″-(4′″-Bromophenyl)-2,2′:6′,2″-terpyridineN,N″-Dioxide (31)

[0159] 3-Chloroperbenzoic acid (29.1 g, 121 mmol) was added to compound30 (12.4 g, 32 mmol) in dichloromethane (500 ml) and the mixture wasstirred overnight at ambient temperature. The mixture was washed with10% sodium carbonate (300 ml), dried (Na₂SO₄) and concentrated.Purification on silica gel (eluent 10% methanol in dichloromethane) gave11.4 g (85%) of product. 1H NMR (dmso-d6) δ: 9.06 (2H, s); 8.43 (2H, m);8.24 (2H, m); 7.80 (4H, s); 7.54 (4H, m). MS (EI+) 419, 421 [M+].

Example 28

[0160] The synthesis of4″-(4′″-Bromophenyl)-2,2′:6′,2″-terpyridine-6,6′-dicarbonitrile (32)

[0161] Trimethylsilylcyanide (13.7 ml, 110 mmol) was added to compound31 (4.6 g, 11 mmol) in dichloromethane (170 ml). After 5 min, benzoylchloride (5.1 ml, 44 mmol) was added within 20 min. Alter stirringovernight, the mixture was evaporated to half volume, 10% solution ofK₂CO₃ (100 ml) was added, the mixture was stirred for 15 min, and theprecipitate filtered and washed with water and cold dichloromethane.Yield was 3.69 g (77%). 1H NMR (dmso-d6): δ 8.98 (2H, d, J 8.0); 8.68(2H, s); 8.31 (2H, t, J 7.6); 8.21 (2H, d, J 7.6); 7.97 (2H, d, J 8.4);7.80 (2H, d, J 8.4). IR (KBr): 2237 cm-1 (CN). MS (EI+)437, 439 [M+].

Example 29

[0162] The synthesis of tetramethyl2,2′,2″,2′″-{[4′-(4″-bromophenyl)-2,2′:6′,2″-terpyridine-6,6″-diyl]bis(methylenenitrilo)}tetrakis(acetate)(34)

[0163] A suspension of compound 32 (3.65 g, 8.3 mmol) in dry THF (100ml) was dearated with argon. BH₃.THF was added during 20 min. Afterstirring for 2.5 h at ice-bath, the excess of borane was destroyed byaddition of methanol. The mixture was evaporated, and the residue wasdissolved in methanol saturated with HCl (50 ml). After stirring for 2 hat room temperature, the mixture was concentrated. The residue wassuspended in THF, filtered, washed with THF and dried. This material wassuspended in dry DMF (50 ml). Diisopropylethylamine (21 ml), methylbromoacetate (3.1 ml, 33.3 mmol) and KI (1.51 g, 9.1 mmol) were added,and the mixture was stirred overnight at room temperature andconcentrated. The residue was dissolved in dichloromethane (80 ml),washed with sat NaHCO₃ (3·40 ml) and dried. Purification was performedon silica gel (eluent pet. ether:ethyl acetate:triethylamine 5:2:1,v/v/v) Yield was 6.6 g. 1H NMR (CDCl₃) δ:8.68 (2H, s); 8.55 (2H, d, J6); 7.87 (2H, t, J 6); 7.81 (2H, d, J 6); 7.68 (2H, d, J 6); 7.62 (2H,d, J 6); 4.19 (4H, s); 3.73 (8H, s); 3.70 (12H, s).

Example 30

[0164] The synthesis of tetramethyl2,2′,2″,2′″-{[4′-(4″-(6-hydroxy-2-hexyn-1-yl)phenyl)-2,2′:6′,2″-terpyridine-6,6″-diyl]bis(methylenenitrilo)}tetrakis(acetate)(35)

[0165] Compound 34 (2.0 g, 2.72 mmol) and 5-hexyn-1-ol (360 ml; 3.28mmol) were dissolved in the mixture of dry THF (15 ml) and triethylamine(4 ml) and the mixture was deaerated with argon for 10 min. Pd(Ph3P)2Cl2(37.5 mg, 0.053 mmol) and CuI (21.9 mg, 0.11 mmol) were added and themixture was stirred overnight at 60° C. The cooled mixture was filteredand the filtrate was concentrated in vacuo. The residue was dissolved indichloromethane (50 ml), washed with water (2·20 ml) and dried.Purification on silica gel (eluent 10% methanol in dichloromethane(v/v)) gave 1.63 g (80%) of product. IR (film) 2232 cm⁻¹ (C≡C, weak). 1HNMR (CDCl₃) δ: 8.70 (2H, s); 8.55 (2H, d, J 7.9); 7.87 (2H, t, J 7.9);7.85 (2H, d, J 8.6); 7.61 (2H, d, J 7.6); 7.56 (2H, d, J 8.2); 4.19 (4H,s); 3.73 (8H, s); 3.75 (2H, m); 3.70 (12H, s); 2.52 (2H, t, 6.7); 1.77(6H, m); 1.74 (1H br). MS (FAB+) 752.

Example 31

[0166] The synthesis of2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-3-(2,2′,2″,2′″-{[4′-(4″-(5-hexyn-6-yl)phenyl)-2,2′:6′,2″-terpyridine-6,6″-diyl]bis(methylenenitrilo)}tetrakis(acetato)uridine(36)

[0167] 2′-Deoxy-5′-O-(4,4′-dimethoxytrityl)uridine was allowed to reactwith compound 35 under Mitsunobu conditions as described for compound 1.Purification was performed on silica gel (eluent petr. ether:ethylacetate: triethylamine; 2:5:1; v/v/v). Yield was 61%. ¹H NMR (CDCl₃) δ:8.70 (2H, s.); 8.55 (2H, d); 7.86 (4H, m); 7.76 (1H, d); 7.57 (4H, m);7.37 (nH, d); 6.83 (4H, d); 6.37 (1H, t); 5.45 (1H, d); 4.59 (1H, m);4.21 (4H, s); 4.09 (1H, m); 3.99 (2H, t); 3.79 (8H, s); 3.70 (12H, s);3.49 (2H, m); 2.79 (1H, br s); 2.53 (2H, m and t); 2.29 (1H, m); 1.78(4H, m).

Example 32

[0168] The synthesis of2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-3-6-{{4-{6,6″-bis[N,N-bis-(methoxycarbonylmethyl)aminomethyl]-2,2′:6′,2″-terpyridine-4′-yl}phenyl}hex-5-yn-1-yl}uridine3′-[O-(2-cyanoethyl)-N,N-diisopropyl]phosphoramidite (37)

[0169] Phosphitylation of compound 36 using the method described inExample 3 yielded the title compound after silica gel columchlromatography as a white powder ³¹P NMR (CDCl₃): δ 148.7 (0.5 P),148.3 (0.5 P).

Example 33

[0170]6-{4-{6,6″-bis[N,N-bis(methoxycarbonylmethyl)aminomethyl]-2,2′:6′,2″-terpyridine-4′-yl}phenyl}hex-5-yn-1-ol[O-(2-cyanoethyl)-N,N-diisopropyl]-phosphoramidite(38).

[0171] Phosphitylation of compound 30 yielded the title compound as aColorless oil (purified on silica gel) ³¹p NMR (CDCl3): δ 147.7 (1 P).

Example 34

[0172] Introduction of Primary Amino Groups to the OligonucletideStructure with the Aid of Compound 3—Labeling of the Amino Groups withan Europium(III) Chelate

[0173] A model sequence d(TTCCTCCACTGT) was synthesized on an ABIinstrument, and 5 phosphoramidites 3 were coupled to its 5′-terminususing standard conditions (concentration 0.1 M in acetonitrile, couplingtime 30 s): No difference in coupling efficiency between 3 and normalnucleosidic building blocks were detected as judged on DMTr-cationresponse. After standard ammoniolytic deprotection, the oligonucleotideprepared was isolated on PAGE and desaltd on NAP columns. Thisoligonucleotide was finally labeled with the non-luminescenteuropium(III) chelate (39) as described in Dahlén, P., Liukkonen, L.,Kwiatkowski, M., Hurskainen, P., Iitiä, A., Siitari, H., Ylikoski, J.,Mukkala, V.-M., and Lövgren, T., Bioconjugate Chem., 1994, 5, 268.

Example 35

[0174] Introduction of Lanthanide(III) Chelates to the OligonucletideStructure with the Aid of Compound 8

[0175] Model sequences were synthesized as described above in Example34. One or 10 phosphoramidites 8 were coupled to its 5′-terminus usingstandard conditions. No difference in coupling efficiency between 8 andnormal nucleosidic building blocks were detected. When the chainassembly was completed, the oligonucleotides were deprotected by firsttreating the solid support with 0.1 M sodium hydroxide for 4 h atambient temperature. 1.0 M ammonium chloride was then added, and thesolution was concentrated in vacuo. The residue was treated with conc.ammonia for 16 h at 60° C., after which europium citrate (10 eq. perligand) was added, and the mixture was kept 90 min at room temperature.Desalting by NAP followed by RP HPLC yielded the desired oligonucleotideconjugates containing one or ten europium(III) chelates in theirstructure.

Example 36

[0176] Introduction of a Lanthanide(III) Chelate to the OligonucleotideStructure with the Aid of Compound 38

[0177] The luminescent terpyridine chelate was introduced to the5′-terminus of the oligonucleotide structure in the aid of blocks 38analogously as described for block 8, except DMTr-On synthesis wasapplied.

1 1 1 12 DNA Unknown Model Sequence 1 ttcctccact gt 12

1-13. (Canceled).
 14. A labeling reactant of formula (I) suitable forlabeling an oligonucleotide

wherein R is a protecting group or is not present; A is eitheraphosphorylating moiety

 where L is O, S, or is not present L′ is H, L′″CH₂CH₂CN or L′″r, whereAr is phenyl or its substituted derivative, where the substituent isnitro or chlorine, and L′″ is O or S; L″ is O⁻, S⁻, Cl, N(i-Pr)₂; or Ais a solid support tethered to Z via a linker arm, which is formed ofone to ten moieties, each moiety being selected from a group consistingof phenylene, alkylene containing 1-12 carbon atoms, ethynediyl, ether,thioether, amide, carbonyl, ester, disulfide, diaza, and tertiary amine;Z is a bridge point and is formed from

 or trivalent derivatives, substituted or unsubstituted, of cyclohexane,cyclohexene, cyclohexadiene, phenyl, cyclopentane, cyclopentene,cyclopentadiene, cyclobutane, cyclobutene, cyclobutadiene, aziridine,diaziridine, oxetane, thietaneazete, azetidine, 1,2-dihydro-1,2-diazete,1,2-diazetidine, furan, tetrahydrofuran, thiophene,2,5-dihydrothiophene, thiolane, selenophene, pyrrole, pyrrolidine,phosphole, 1,3-dioxolane, 1,2-dithiole, 1,2-thiolane, 1,3-dithiole,1,3-dithiolane, oxazole, 4,5-dihydrooxazole, isoxazole,4,5-dihydoisozaole, 2,3-dihydroisoxazole, thiazole, isothiazole,imidazole, imidazolidine, pyrazole, 4,5-dihydropyrazole, pyrazolidine,triazole, pyran, pyran-2-one, 3,4-dihydro-2H-pyran, tetrahydropyran,4H-pyran, pyran-4-one, pyridine, pyridone, piperidine, phosphabenzene,1,4-dioxin, 1,4-dithiin, 1,4-oxathiin, oxazine, 1,3-oxazinone,morpholine, 1,3-dioxane, 1,3-dithiane, pyridazine, pyrimidine, pyrazine,piperazine, 1,2,4-triazine, 1,3,5-triazine,1,3,5-triaza-cyclohexane-2,4,6-trione; where R″ is H or X′X″, where X′is —O—, —S—, —N—, ON— or —NH— and X″ is a protection group or X′ is —O—and X″ is alkyl or alkoxyalkyl; X is H, alkyl, alkynyl, allyl, Cl, Br,I, F, S, O, NHCOCH(CH₃)₂, NHCOCH₃, NHCOPh, SPh₃, OCOCH₃ or OCOPh; E is alinker arm between R and Z and is formed of one to ten moieties, eachmoiety being selected from a group consisting of phenylene, alkylenecontaining 1-12 carbon atoms, ethynediyl, ether, thioether, amide,carbonyl, ester, disulfide, diaza, and tertiary amine, or not present;E′ is a linker arm between G and Z, and is formed of one to tenmoieties, each moiety being selected from the group consisting ofphenylene, alkylene containing 1-12 carbon atoms, ethynediyl, ether,thioether, amide, carbonyl, ester, disulfide, diaza, and tertiary amine,or is not present; G is a bivalent aromatic structure, tethered to twoiminodiacetic acid ester groups N(CH₂COOR′″)₂ where R′″ is an alkyl of 1to 4 carbon atoms, allyl, ethyltrimethylsilyl, phenyl or benzyl, whichphenyl or benzyl is substituted or unsubstituted, and said bivalentaromatic structure is capable of absorbing light or energy andtransferring the excitation energy to a lanthanide ion after the solidphase synthesis made labeling reactant has been released from the usedsolid support, deprotected and converted to a lanthanide chelate, or Gis a structure selected from a group consisting of

 where R′″ is an alkyl of 1 to 4 carbon atoms, allyl,ethyltrimethylsilyl, phenyl or benzyl, which phenyl or benzyl issubstituted or unsubstituted, and one of the hydrogen atoms issubstituted with E′, or G is a protected functional group, where thefunctional group is amino, aminooxy, carboxyl, thiol, and the protectinggroup is phthaloyl, trityl, 2-(4-nitrophenylsulfonyl)ethoxycarbonyl,fluorenylmethyloxycarbonyl, benzyloxycarbonyl, trifluoroacetyl ort-butoxycarbonyl for amino and aminooxy, alkyl for carbonyl and alkyl ortrityl for thiol, provided that bridge point Z is selected from a groupconsisting of


15. The labeling reactant of claim 14, wherein R is a member of thegroup consisting of 4,4′dimethoxytrityl, 4-methoxytrityl, trityl, and(9-phenyl)xanthen-9-yl.
 16. The labeling reactant of claim 14, whereinX″ is a member of the group consisting of t-butyldimethylsilyl-,tetrahydropyranyl, 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl-,1-[2-chloro-4-methyl)phenyl]-4-metoxypiperidin-4-yl-,4-methoxytetrahydropyran-4-yl-, phthaloyl-, acetyl, pivaloyl-, benzoyl-,4-methylbenzoyl, benzyl-, and trityl.
 17. The labeling reactant of claim14, wherein G is a protected functional group.
 18. The labeling reactantof claim 17, wherein said protected functional group is selected fromthe group consisting of amino, carboxyl, aminooxy and thiol.
 19. Thelabeling reactant of claim 14, wherein G is an organic dye.
 20. Thelabeling reactant of claim 19, wherein said organic dye is selected fromthe group consisting of dabsyl, dansyl, fluorescein, rhodamine andtetramethyl-6-carboxyrhodamine (TAMRA).
 21. The labeling reactant ofclaim 14, wherein the temporary protecting group R is4,4′-dimethoxytrityl.
 22. The labeling reactant of claim 14, whereinsaid reactant is a nucleotide and the sugar of the nucleotide is2-deoxyribose or 3-deoxyribose.
 23. The labeling reactant of claim 22,wherein X′ is hydroxyl.
 24. The labeling reactant of claim 23, whereinthe permanent protection group X″ of X′ is selected from the groupconsisting of t-butyldimethylsilyl, tetrahydropyranyl,1-(2-fluorophenyl)-4-methoxypiperidin-4-yl-,1-[2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl- and4-methoxytetrahydropyran-4-yl-.
 25. The labeling reactant of claim 22,wherein X″ is an alkyl or alkoxyalkyl.
 26. The labeling reactant ofclaim 25, wherein X″ is selected from the group consisting of methyl,methoxymethyl and ethoxymethyl.
 27. The labeling reactant of claim 14,wherein G is a bivalent aromatic structure.
 28. The labeling reactant ofclaim 27, wherein G is selected from the group consisting ofcarbostyryl,


29. The labeling reactant of claim 14, wherein said reactant isnon-luminescent and G is selected from a group consisting of

and wherein R′″ is an alkyl of 1 to 4 carbon atoms, allyl,ethyltrimethylsilyl, phenyl or benzyl, which phenyl or benzyl can besubstituted or unsubstituted, and one of the hydrogen atoms issubstituted with E′.
 30. The labeling reactant of claim 29, wherein R′″is selected from the group consisting of methyl, ethyl and allyl. 31.The labeling reactant of claim 14 wherein A is a solid support.
 32. Thelabeling reactant of claim 31, where A is5′-O-(4,4′-dimethoxytrityl)-3′-O-succinyl-N3-{tetramethyl-2,2′,2″,2′″-{6,6′-[4′-hydroxyethoxyethoxyphenylethynyl]pyridine-2,6-diyl}bis-(methylenenitrilo)tetrakis(acetato)}thymidinelong chain alkylamine controlled pore glass.
 33. A method for a directattachment of a conjugate group to an oligonucleotide structure enablingthe attachment of a desired number of these groups during chain assemblywherein it comprises a Mitsunobu alkylation of a compound of formula(II) R-Z  (II) wherein R is a protecting group; Z′ is an acidic bridgepoint selected from a group consisting of

 where R″ is H or X′X″, where X′ is —O—, —S—, —N—, ON— or —NH— and X″ isa protection group; X is H, alkyl, alkynyl, allyl, Cl, Br, I, F, S, O,NHCOCH(CH₃)₂, NHCOCH₃, NHCOPh, SPh₃, OCOCH₃ or OCOPh; and pK_(a) of saidacidic bridge point is <14; with a compound of formula (III) G-E″  (III) wherein E″ is an arm with a primary aliphatic OH group at the end,which arm is formed of one to ten moieties, each moiety being selectedfrom a group consisting of phenylene, alkylene containing 1-12 carbonatoms, ethynediyl, ether, thioether, amide, carbonyl, ester, disulfide,diaza, and tertiary amine; G is a bivalent aromatic structure, tetheredto two iminodiacetic acid ester groups N(CH₂COOR′″)₂, where R′″ is analkyl of 1 to 4 carbon atoms, allyl, ethyltrimethylsilyl, phenyl orbenzyl, which phenyl or benzyl can be substituted or unsubstituted andsaid bivalent aromatic structure is capable of absorbing light or energyand transferring the excitation energy to a lanthanide ion after thesolid phase synthesis made labeling reactant has been released from theused solid support, deprotected and converted to a lanthanide chelate,or G is a structure selected from a group consisting of

 where R′″ is an alkyl of 1 to 4 carbon atoms, allyl,ethyltrimethylsilyl, phenyl or benzyl, which phenyl or benzyl can besubstituted or unsubstituted, and one of the hydrogen atoms issubstituted with E′, or G is a protected functional group, where thefunctional group is amino, aminooxy, carboxyl, thiol, and the protectinggroup is pthaloyl, trityl, 2-(4-nitrophenyl-sulfonyl)ethoxycarbonyl,fluorenylmethyloxycarbonyl, benzyloxycarbonyl or t-butoxycarbonyl foramino and aminooxy, alkyl for carbonyl and alkyl or trityl for thiol, orG is not present; and the functional groups of E′ and G, excluding saidprimary aliphatic OH group, are protected; to produce a compound offormula (IV)

 wherein G and R of compound (IV) are as defined above; E′″ is a linkerarm between G and Z, and is formed of one to ten moieties, each moietybeing selected from a group consisting of phenylene, alkylene containing1-12 carbon atoms, ethynediyl, ether, thioether, amide, carbonyl, ester,disulfide, diaza, and tertiary amine; and Z″ is a bridge point selectedfrom a group consisting of

 where R″ is H or X′X″, where X′ is —O—, —S—, —N—, ON— or —NH— and X″ isa protection group; X is H, alkyl, alkynyl, allyl, Cl, Br, I, F, S, O,NHCOCH(CH₃)₂, NHCOCH₃, NHCOPh, SPh₃, OCOCH₃ or OCOPh.
 34. The method ofclaim 33, wherein X″ is a member selected from the group consisting oft-butyldimethylsilyl-, tetrahydropyranyl,1-(2-fluorophenyl)-4-methoxypiperidin-4-yl-,1-[2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl-,4-methoxytetrahydropyran-4-yl-, pthaloyl-, acetyl, pivaloyl-, benzoyl-,4-methylbenzoyl, benzyl-, trityl and alkyl.